DNA expansions generated by human Polμ on iterative sequences.

Abstract

Polµ is the only DNA polymerase equipped with template-directed and terminal transferase activities. Polµ is also able to accept distortions in both primer and template strands, resulting in misinsertions and extension of realigned mismatched primer terminus. In this study, we propose a model for human Polµ-mediated dinucleotide expansion as a function of the sequence context. In this model, Polµ requires an initial dislocation, that must be subsequently stabilized, to generate large sequence expansions at different 5'-P-containing DNA substrates, including those that mimic non-homologous end-joining (NHEJ) intermediates. Our mechanistic studies point at human Polµ residues His(329) and Arg(387) as responsible for regulating nucleotide expansions occurring during DNA repair transactions, either promoting or blocking, respectively, iterative polymerization. This is reminiscent of the role of both residues in the mechanism of terminal transferase activity. The iterative synthesis performed by Polµ at various contexts may lead to frameshift mutations producing DNA damage and instability, which may end in different human disorders, including cancer or congenital abnormalities.

Polµ generates a large sequence expansion on a DNA gap with a specific trinucleotide sequence. In the scheme, the template sequence indicated (NNN) corresponds to the trinucleotides shown below. Subindex ‘2’ indicates that each trinucleotide sequence is twice repeated at the gap. Polymerization reactions (described in ‘Materials and Methods’ section) were performed in the presence of 4 nM of the indicated DNA substrate in each case, 2 mM MgCl2, 270 nM of each polymerase and 50 µM mix dNTPs (using the complementary nucleotides to the gap sequence in each case). After 1 h incubation at 30°C, polymerization products were analysed by electrophoresis on 20% polyacrylamide/8 M urea gels and autoradiography. P indicates the unextended primer; +6 the elongated product upon complete gap-filling; +18 the fully elongated product after gap-filling and strand displacement; the products of nucleotide expansion are indicated with a bracket.

A repeated nucleotide at the end of a gap is required to generate large sequence expansions. In the scheme, the template sequence indicated (NNN) corresponds to the trinucleotides shown below, that could be present twice (subindex 2) or only once (subindex 1). Polymerization reactions (described in ‘Materials and Methods’ section) were performed in the presence of 4 nM of the indicated DNA substrate in each case, 2 mM MgCl2, 270 nM of either Polλ or Polµ and 50 µM mix dNTPs (using the complementary nucleotides to the gap sequence in each case). After 1 h incubation at 30°C, polymerization products, including sequence expansions, were analysed by electrophoresis on 20% polyacrylamide/8 M urea gels and autoradiography.

A distortion upstream to the dinucleotide is a prerequisite for the formation of Polµ-mediated sequence expansions during gap-filling. (A) In the scheme, the template sequence indicated (CNN) corresponds to the four trinucleotides shown below. The first base at the trinucleotide is always dC, followed by the four different homo-dinucleotides. Polymerization reactions (described in ‘Materials and Methods’ section) were performed in the presence of 4 nM of the indicated DNA substrate in each case, 2 mM MgCl2, 270 nM of either Polλ or Polµ and 50 µM mix dNTPs (using the complementary nucleotides to the gap sequence in each case). (B) Importance of the first nucleotide of the triplet (NAA) for the efficiency of expansion by Polμ. The gap sequence used and the different combinations of nucleotides provided, were as indicated. Polymerization reactions were performed as in (A). After 1 h incubation at 30°C, polymerization products were analysed by electrophoresis on 20% polyacrylamide/8 M urea gels and autoradiography.

His329 allows while Arg387 limits sequence expansions by Polµ. (A) Top: Model of Polµ bound to a template/primer substrate in which the 3′-protruding primer terminus is in an unproductive position, occupying the incoming dNTP site. Residue Arg387, in a conformation modelled to match that of the lysine present in TdT in a similar structure, is contacting the primer impeding its backwards translocation. His329, modelled in the conformation observed for the same residue in the crystal of TdT bound to an ssDNA primer, is not making any contacts with the DNA substrate. Bottom: Crystal structure of Polµ bound to a gapped DNA substrate and incoming dNTP. His329 has rotated and is contacting both incoming dNTP and primer terminus, helping to reposition the latter. Arg387 is now contacting the template strand (n − 3 position; indicated in yellow), having allowed the movement backwards of the primer. DNA substrates are indicated in dark (primer strand) and light (template and downstream strand) blue. Incoming dNTP is indicated in green. (B) In the scheme, the template sequence indicated (NAA) corresponds to the four trinucleotides shown below. The two last bases always form the dinucleotide AA, preceded by any of the four different nucleotides. Polymerization reactions (described in Materials and Methods section) were performed in the presence of 4 nM of the indicated DNA substrate in each case, 2 mM MgCl2, 270 nM Polµ and 50 µM mix dNTPs (using the complementary nucleotide to the dinucleotide AA). After 1 h incubation at 30°C, polymerization products were analysed by electrophoresis on 20% polyacrylamide/8 M urea gels and autoradiography.

Impact of the generation of sequence expansions during NHEJ repair reactions by Polµ. (A) The scheme corresponds to the end-joining substrates used, whose 3′-protrusions can be connected by three bases pairs but leaving a distortion (1 flipped-out base) close to the 3′-primer terminus. Such a connection leaves two different 1-nt gaps. Gap-filling of one of them (that flanked by a 5′-P) is evaluated as a function of each possible templating base (X). Thus, the 5′-labelled substrate (dark grey) will be tested as primer, whereas the cold substrate (light grey; in which the X in the scheme is changed to A, C, G or T) is providing the template for the connection. Polymerization reactions were performed in the presence of 200 nM Polµ, 2.5 mM MgCl2 and 100 µM of a single dNTP (complementary to X in each case). After incubation for 1 h at 30°C, reactions were stopped and loaded on 20% PA-8 M urea gels. Labelled DNA fragments were detected by autoradiography. (B) Polymerization reactions performed as in (A), but using end-joining substrates used whose 3′-protrusions can be connected by three bases pairs with no distortion.

Mechanistic model for dinucleotide expansions generated by human Polµ. Thick arrows indicate more efficient reactions, whereas thin arrows are related to slower or less favourable reactions. (A) Trinucleotide conformed by the same nucleotide. In this case, no distortion associated to gap-filling is generated, thus precluding a large expansion. (B) After polymerase binding and realignment of the primer terminus, a ‘slippage-mediated’ dislocation is formed, creating a template distortion. In this situation, a large expansion reaction is observed. (C) In this case the distortion is induced by a ‘dNTP-selection-mediated’ dislocation of the template strand, again resulting in the generation of large sequence expansions. (D) In a NHEJ context, a repeated nucleotide neighbour to the 5′-P can induce nucleotide expansions by Polµ, although in this case, a pre-existing stable distortion or impairing is not strictly required.